Several pathological conditions, such as diabetes mellitus and tumor cell metastasis, require interactions of proteins, glycosaminoglycans (GAGs), or cells with the triple-helical regions of native collagens. For example, tumor cell metastasis involves the adhesion and motility of tumor cells on extracellular matrix components such as laminin and collagen. The relative importance of collagen primary, secondary, and tertiary structure on these interactions has not been ascertained. To elucidate the roles of collagen structures on protein, GAG, and cell activities, single-stranded and triple-helical synthetic peptides need to be studied. To accomplish this, a general synthetic peptide methodology needs to be developed by which the importance of collagen triple-helical structure on biological activities can be evaluated. This methodology should also allow for incorporation of post-translationally modified amino acids, e.g. glycosylated amino acid residues, as types II and IV collagen are glycosylated.
Collagens are composed of three .alpha. chains of primarily repeating Gly-X-Y triplets, which induces each .alpha. chain to adopt a left-handed polyPro II helix. Three left-handed chains then intertwine to form a right-handed super-helix. Homotrimeric collagens (i.e., types II and III) have three .alpha. chains of identical sequence, whereas heterotrimeric collagens have two .alpha. chains of identical sequence (designated .alpha.1) and one .alpha. chain of differing sequence (designated .alpha.2) (i.e., type I) or three .alpha. chains of differing sequence (designated .alpha.1, .alpha.2, and .alpha.3) (i.e., type VI). Homotrimeric triple-helical polypeptides of defined molecular weight were created initially by solid-phase incorporation of tertiary-amyloxycarbonyl-X-Y-Gly tripeptides (prepared in solution), hydrogen fluoride cleavage, and interchain association in aqueous acetic acid. See, for example, S. Sakakibara et al., Bull. Chem. Soc. Jpn. 41, 1273 (1968). The most thermally stable triple-helices were formed with repeating Pro-Pro-Gly or Pro-Hyp-Gly triplets, with increased thermal stability resulting from Hyp versus Pro in the Y position. See, for example, J. Engel et al., Biopolymers 16, 601 (1977) and R. W. Webber et al., Helv. Chim. Acta 61,701 (1978).
To more fully understand the subtleties of collagen structure, it is desirable to insert sequences other than Gly-Pro-Pro or Gly-Pro-Hyp within a triple-helix and correlate the effects of these sequences on triple-helical structure and biological activity. Such sequences should be aligned in the triple-helical peptide as they would be in native collagens. To ensure proper alignment of three peptide strands in a triple-helix, a branching protocol was developed for liquid-phase peptide synthesis by Heidemann and coworkers. See, for example, W. Roth et al., Makromol. Chem. 180, 905 (1979) and H.-P. Germann et at., Biopolymers 27, 157 (1988). The branch was introduced at the C-terminus of the synthetic peptide, consistent with the natural nucleation of collagen triple-helices from the C- to the N-terminus. A variation of this branching protocol was developed for solid-phase synthesis. See, for example, G. B. Fields et al., in Innovation and Perspectives in Solid Phase Synthesis (R. Epton, Ed.), pp. 241-260, Solid Phase Conference Coordination Ltd., Birmingham, U.K. (1990).
Such liquid- and solid-phase methodologies do not allow for the incorporation of glycosylated residues, as O-glycosidic bonds are not stable to repetitive moderate acidolysis and strong acid cleavage conditions. Glycosylated 5-hydroxy-L-lysine (Hyl) residues are found in regions of collagen-mediated biological activities such as cell adhesion and migration and heparin binding. Glycosylation also effects protein secondary structure, inducing .beta.-turns in single-stranded peptides. Solid-phase peptide synthesis utilizing base-labile 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids has become increasingly popular due to the fairly mild chemical conditions employed, which permit efficient incorporation of glycosylated residues. See, for example, G. B. Fields et at., Int. J. Peptide Protein Res. 35, 161 (1990). In addition, other acid labile residues, such as Trp or .sup.2 H-labeled amino acids (for NMR studies), are more efficiently incorporated by Fmoc chemistry than standard tertiary-butyloxycarbonyl (Boc) chemistry. Recent advances in Fmoc chemistry, including the development of three-dimensional orthogonal schemes, has permitted the design of synthetic protocols for the mild solid-phase synthesis of branched, triple-helical peptides. Triple-helical peptides synthesized under these mild conditions are far less likely to be contaminated by by-products as well as potentially incorporating the greatest variety of unusual and non-native amino acid residues.